Cell Wall Epitopes and Endoploidy as Reporters of Embryogenic Potential in Brachypodium Distachyon Callus Culture.

Alexander Betekhtin,Magdalena Rojek,Robert Hasterok,Katarzyna Nowak,Ewa Kurczynska,Artur Pinski,Anna Milewska-Hendel,John H Doonan

doi:10.3390/ijms19123811

Abstract

Effective regeneration of callus tissue into embryos and then into whole plants is essential for plant biotechnology. The embryonic potential is often low and can further decrease with time in culture, which limits the utilisation of calli for transformation procedures and in vitro propagation. In this study, we show that the loss of embryogenic potential in callus cultures of Brachypodium distachyon is progressive over time. Flow cytometry analyses indicated endoploidy levels increased in 60- and 90-day-old calli with effective loss of the 2C DNA content peak in the latter. Analysis of indolic compounds content revealed a decrease in 60- and 90-day-old calli compared to either freshly isolated explants or 30-day-old calli. Immunohistochemical analysis revealed a decrease in arabinogalactan proteins (AGP) signal with the time of culture, but extensin (EXT) epitopes either increased (JIM12 epitopes) or decreased (JIM11 epitopes). The transcript accumulation levels of AGPs and EXTs confirmed these results, with most of AGP and EXT transcripts gradually decreasing. Some chimeric EXT transcripts significantly increased on the 30th day of culture, perhaps because of an increased embryogenic potential. Selected somatic embryogenesis-related genes and cyclins demonstrated a gradual decrease of transcript accumulation for YUCCA (YUC), AINTEGUMENTA-LIKE (AIL), BABY BOOM (BBM), and CLAVATA (CLV3) genes, as well as for most of the cyclins, starting from the 30th day of culture. Notably, WUSCHEL (WUS) transcript was detectable only on the 30th and 60th day and was not detectable in the zygotic embryos and in 90-day-old calli.

Highlights

Somatic embryogenesis (SE) occurs when a single somatic cell or group of somatic cells, usually induced by external conditions of in vitro culture, changes its developmental programme and starts forming totipotent embryogenic cells capable of becoming complete plants [1]
The correct progression of SE is controlled by a large group of transcription factors, e.g., BABY BOOM (BBM), WUSHEL (WUS), LEAFY COTYLEDON1 (LEC1) and LEAFY COTYLEDON2 (LEC2), FUSCA3 (FUS3), proteins associated with signal transduction pathways, among which major groups are SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK proteins) and CLAVATA proteins (CLV1, 2 and 3), as well as proteins involved in the regulation of hormone biosynthesis and response, for example, YUCCA1 (YUC1)
The eukaryotic cell cycle is controlled by the large family of CYCLIN-DEPENDENT KINASES (CDKs)

Summary

Introduction

Somatic embryogenesis (SE) occurs when a single somatic cell or group of somatic cells, usually induced by external conditions of in vitro culture, changes its developmental programme and starts forming totipotent embryogenic cells capable of becoming complete plants [1]. The correct progression of SE is controlled by a large group of transcription factors, e.g., BABY BOOM (BBM), WUSHEL (WUS), LEAFY COTYLEDON1 (LEC1) and LEAFY COTYLEDON2 (LEC2), FUSCA3 (FUS3), proteins associated with signal transduction pathways, among which major groups are SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK proteins) and CLAVATA proteins (CLV1, 2 and 3), as well as proteins involved in the regulation of hormone biosynthesis and response, for example, YUCCA1 (YUC1) These proteins are involved, variously, in dedifferentiation, promotion, and maintenance of cell totipotency and embryogenic cell formation [1,2,3,4,5]. The activity of these kinases is positively regulated by binding to cyclins, some of which appear and disappear periodically during the cell cycle, and their gene expression is often modulated during the progression of SE [7,8]

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